Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface.

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.

Neutrophilia in severe asthma is reduced in Ormdl3 overexpressing mice.

Genome-wide association studies have linked the ORM (yeast)-like protein isoform 3 (ORMDL3) to asthma severity. Although ORMDL3 is a member of a family that negatively regulates serine palmitoyltransferase (SPT) and thus biosynthesis of sphingolipids, it is still unclear whether ORMDL3 and altered sphingolipid synthesis are causally related to non-Th2 severe asthma associated with a predominant neutrophil inflammation and high interleukin-17 (IL-17) levels. Here, we examined the effects of ORMDL3 overexpression in a preclinical mouse model of allergic lung inflammation that is predominantly neutrophilic and recapitulates many of the clinical features of severe human asthma. ORMDL3 overexpression reduced lung and circulating levels of dihydrosphingosine, the product of SPT. However, the most prominent effect on sphingolipid levels was reduction of circulating S1P. The LPS/OVA challenge increased markers of Th17 inflammation with a predominant infiltration of neutrophils into the lung. A significant decrease of neutrophil infiltration was observed in the Ormdl3 transgenic mice challenged with LPS/OVA compared to the wild type and concomitant decrease in IL-17, that plays a key role in the pathogenesis of neutrophilic asthma. LPS decreased survival of murine neutrophils, which was prevented by co-treatment with S1P. Moreover, S1P potentiated LPS-induced chemotaxis of neutrophil, suggesting that S1P can regulate neutrophil survival and recruitment following LPS airway inflammation. Our findings reveal a novel connection between ORMDL3 overexpression, circulating levels of S1P, IL-17 and neutrophil recruitment into the lung, and questions the potential involvement of ORMDL3 in the pathology, leading to development of severe neutrophilic asthma.

Cellular inhibitor of apoptosis 2 (cIAP2) restricts neuroinflammation during experimental autoimmune encephalomyelitis.

BACKGROUND: Immune activation, neuroinflammation, and cell death are the hallmarks of multiple sclerosis (MS), which is an autoimmune demyelinating disease of the central nervous system (CNS). It is well-documented that the cellular inhibitor of apoptosis 2 (cIAP2) is induced by inflammatory stimuli and regulates adaptive and innate immune responses, cell death, and the production of inflammatory mediators. However, the impact of cIAP2 on neuroinflammation associated with MS and disease severity remains unknown. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS, to assess the effect of cIAP2 deletion on disease outcomes. We performed a detailed analysis on the histological, cellular, and molecular levels. We generated and examined bone-marrow chimeras to identify the cIAP2-deficient cells that are critical to the disease outcomes. RESULTS: cIAP2-/- mice exhibited increased EAE severity, increased CD4+ T cell infiltration, enhanced proinflammatory cytokine/chemokine expression, and augmented demyelination. This phenotype was driven by cIAP2-deficient non-hematopoietic cells. cIAP2 protected oligodendrocytes from cell death during EAE by limiting proliferation and activation of brain microglia. This protective role was likely exerted by cIAP2-mediated inhibition of the non-canonical NLRP3/caspase-8-dependent myeloid cell activation during EAE. CONCLUSIONS: Our findings suggest that cIAP2 is needed to modulate neuroinflammation, cell death, and survival during EAE. Significantly, our data demonstrate the critical role of cIAP2 in limiting the activation of microglia during EAE, which could be explored for developing MS therapeutics in the future.

Ceramide in apoptosis and oxidative stress in allergic inflammation and asthma.

BACKGROUND: Nothing is known about the mechanisms by which increased ceramide levels in the lung contribute to allergic responses and asthma severity. OBJECTIVE: We sought to investigate the functional role of ceramide in mouse models of allergic airway disease that recapitulate the cardinal clinical features of human allergic asthma. METHODS: Allergic airway disease was induced in mice by repeated intranasal administration of house dust mite or the fungal allergen Alternaria alternata. Processes that can be regulated by ceramide and are important for severity of allergic asthma were correlated with ceramide levels measured by mass spectrometry. RESULTS: Both allergens induced massive pulmonary apoptosis and also significantly increased reactive oxygen species in the lung. Prevention of increases in lung ceramide levels mitigated allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. In contrast, dietary supplementation of the antioxidant α-tocopherol decreased reactive oxygen species but had no significant effects on elevation of ceramide level or apoptosis, indicating that the increases in lung ceramide levels in allergen-challenged mice are not mediated by oxidative stress. Moreover, specific ceramide species were altered in bronchoalveolar lavage fluid from patients with severe asthma compared with in bronchoalveolar lavage fluid from individuals without asthma. CONCLUSION: Our data suggest that elevation of ceramide level after allergen challenge contributes to the apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide might be the trigger of formation of Creola bodies found in the sputum of patients with severe asthma and could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.

Stat5B is required for IgE-Mediated mast cell function in vitro and in vivo.

Mast cells are found primarily at interfaces with the external environment, where they provide protection from pathogens but also elicit allergic inflammation. Mast cell activation by antigen-induced aggregation of IgE bound to the high affinity receptor, FcεRI, is a critical factor leading to inflammation and bronchoconstriction. We previously found that Stat5 is activated by FcεRI and that Stat5B suppression decreased IgE-induced cytokine production in vitro, but in vivo responses have not been assessed. We now show that Stat5B-deficient (KO) mice have reduced responses to IgE-mediated anaphylaxis, despite normal mast cell tissue distribution. Similarly, Stat5B KO mast cells have diminished IgE-induced degranulation and cytokine secretion in vitro. These mice have elevated IgE production that is not correlated with an intrinsic B cell defect. The current work demonstrates that the Stat5B isoform is required for normal mast cell function and suggests it limits IgE production in vivo.

B Cell ADAM10 Controls Murine Lupus Progression through Regulation of the ICOS:ICOS Ligand Axis.

The role of ICOS and its ligand (ICOSL) have both been shown to be essential for proper humoral responses as well as autoimmune Ab development in mouse models of lupus. In this paper, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using B6mir146a-/- mice and a model of lymphoproliferative disease using the well-characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared with control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA Ab production. In line with this, we found a significant reduction in follicular helper T cells and germinal center B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only show a role of B cell ADAM10 in control autoimmunity but also increase our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity.

The Il9 CNS-25 Regulatory Element Controls Mast Cell and Basophil IL-9 Production.

IL-9 is an important mediator of allergic disease that is critical for mast cell-driven diseases. IL-9 is produced by many cell types, including T cells, basophils, and mast cells. Yet, how IL-9 is regulated in mast cells or basophils is not well characterized. In this report, we tested the effects of deficiency of a mouse Il9 gene regulatory element (Il9 CNS-25) in these cells in vivo and in vitro. In mast cells stimulated with IL-3 and IL-33, the Il9 CNS-25 enhancer is a potent regulator of mast cell Il9 gene transcription and epigenetic modification at the Il9 locus. Our data show preferential binding of STAT5 and GATA1 to CNS-25 over the Il9 promoter in mast cells and that T cells and mast cells have differing requirements for the induction of IL-9 production. Il9 CNS-25 is required for IL-9 production from T cells, basophils, and mast cells in a food allergy model, and deficiency in IL-9 expression results in decreased mast cell expansion. In a Nippostrongylus brasiliensis infection model, we observed a similar decrease in mast cell accumulation. Although decreased mast cells correlated with higher parasite egg burden and delayed clearance in vivo, T cell deficiency in IL-9 also likely contributes to the phenotype. Thus, our data demonstrate IL-9 production in mast cells and basophils in vivo requires Il9 CNS-25, and that Il9 CNS-25-dependent IL-9 production is required for mast cell expansion during allergic intestinal inflammation.

Immunization against Anaplasma phagocytophilum Adhesin Binding Domains Confers Protection against Infection in the Mouse Model.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.

The DNA methyltransferase inhibitor, guadecitabine, targets tumor-induced myelopoiesis and recovers T cell activity to slow tumor growth in combination with adoptive immunotherapy in a mouse model of breast cancer.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) present a significant obstacle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous groups, including our own, have reported on the myelo-depletive effects of certain chemotherapy agents. We have shown previously that decitabine increased tumor cell Class I and tumor antigen expression, increased ability of tumor cells to stimulate T lymphocytes, depleted tumor-induced MDSC in vivo and augmented immunotherapy of a murine mammary carcinoma. RESULTS: In this study, we expand upon this observation by testing a next-generation DNA methyltransferase inhibitor (DNMTi), guadecitabine, which has increased stability in the circulation. Using the 4 T1 murine mammary carcinoma model, in BALB/cJ female mice, we found that guadecitabine significantly reduces tumor burden in a T cell-dependent manner by preventing excessive myeloid proliferation and systemic accumulation of MDSC. The remaining MDSC were shifted to an antigen-presenting phenotype. Building upon our previous publication, we show that guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. We also show guadecitabine's versatility with similar tumor reduction and augmentation of immunotherapy in the C57BL/6 J E0771 murine breast cancer model. CONCLUSIONS: Guadecitabine depleted and altered MDSC, inhibited growth of two different murine mammary carcinomas in vivo, and augmented immunotherapeutic efficacy. Based on these findings, we believe the immune-modulatory effects of guadecitabine can help rescue anti-tumor immune response and contribute to the overall effectiveness of current cancer immunotherapies.

B cell ADAM17 controls T cell independent humoral immune responses through regulation of TACI and CD138.

ADAM17 is known to contribute to the immune system through its shedding of tumor necrosis factor alpha (TNFα). However, the role of ADAM17 in B cell biology is not well characterized. To determine whether B cell ADAM17 contributes to T cell-independent humoral immune responses, we crossed CD19 Cre transgenic mice with mice bearing a floxed allele of ADAM17 (ADAM17CD19). In this study, we show a B cell intrinsic role for ADAM17 in regulating marginal zone B cell (MZB) numbers in mice. Interestingly, we demonstrate that the loss of B cell ADAM17 results in reduced MZB numbers in the naïve state and after immunization with T-independent antigen, yet enhanced humoral immunity to T cell independent antigens. We additionally find elevated TACI and CD138 levels on plasma cells following immunization in ADAM17CD19 mice. Overall, these findings suggest that B cell ADAM17 may orchestrate T independent immune responses through both MZB numbers and plasma cell antibody production.

Fluvastatin Induces Apoptosis in Primary and Transformed Mast Cells.

Statin drugs are widely employed in the clinic to reduce serum cholesterol. Because of their hydroxymethylglutaryl coenzyme A reductase antagonism, statins also reduce isoprenyl lipids necessary for the membrane anchorage and signaling of small G-proteins in the Ras superfamily. We previously found that statins suppress immunoglobulin E (IgE)-mediated mast cell activation, suggesting these drugs might be useful in treating allergic disease. Although IgE-induced function is critical to allergic inflammation, mast cell proliferation and survival also impact atopic disease and mast cell neoplasia. In this study, we describe fluvastatin-mediated apoptosis in primary and transformed mast cells. An IC50 was achieved between 0.8 and 3.5 µM in both cell types, concentrations similar to the reported fluvastatin serum Cmax value. Apoptosis was correlated with reduced stem cell factor (SCF)-mediated signal transduction, mitochondrial dysfunction, and caspase activation. Complementing these data, we found that p53 deficiency or Bcl-2 overexpression reduced fluvastatin-induced apoptosis. We also noted evidence of cytoprotective autophagy in primary mast cells treated with fluvastatin. Finally, we found that intraperitoneal fluvastatin treatment reduced peritoneal mast cell numbers in vivo These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases. SIGNIFICANCE STATEMENT: Fluvastatin, a statin drug used to lower cholesterol, induces apoptosis in primary and transformed mast cells by antagonizing protein isoprenylation, effectively inhibiting stem cell factor (SCF)-induced survival signals. This drug may be an effective means of suppressing mast cell survival.

The Obligate Intracellular Bacterium Orientia tsutsugamushi Targets NLRC5 To Modulate the Major Histocompatibility Complex Class I Pathway.

Orientia tsutsugamushi is an obligate intracellular bacterium that infects mononuclear and endothelial cells to cause the emerging global health threat scrub typhus. The ability of O. tsutsugamushi to survive in monocytes facilitates bacterial dissemination to endothelial cells, which can subsequently lead to several potentially fatal sequelae. As a strict intracellular pathogen that lives in the cytoplasm of host cells, O. tsutsugamushi has evolved to counter adaptive immunity. How the pathogen does so and the outcome of this strategy in monocytes versus endothelial cells are poorly understood. This report demonstrates that O. tsutsugamushi reduces cellular levels of NOD-, LRR-, and CARD-containing 5 (NLRC5), a recently identified specific transactivator of major histocompatibility complex class I (MHC-I) component gene expression, to inhibit MHC-I biosynthesis. Importantly, the efficacy of this approach varies with the host cell type infected. In nonprofessional antigen-presenting HeLa and primary human aortic endothelial cells, the O. tsutsugamushi-mediated reduction of NLRC5 results in lowered MHC-I component transcription and, consequently, lower total and/or surface MHC-I levels throughout 72 h of infection. However, in infected THP-1 monocytes, which are professional antigen-presenting cells, the reductions in NLRC5 and MHC-I observed during the first 24 h reverse thereafter. O. tsutsugamushi is the first example of a microbe that targets NLRC5 to modulate the MHC-I pathway. The differential ability of O. tsutsugamushi to modulate this pathway in nonprofessional versus professional antigen-presenting cells could influence morbidity and mortality from scrub typhus.

A Disintegrin and Metalloproteinase 17 is required for ILC2 responses to IL-33.

Group 2 innate lymphoid cells (ILC2s) play an important role in the initiation of type-2 immune responses. Numerous targets have been identified that may activate or repress ILC2 function, though few negative regulatory feedback pathways induced upon activation have been shown to be operative in ILC2s. Here we demonstrate that loss of ADAM17 from ILC2s results in a selective defect in IL-33 responsiveness, but not IL-25 responsiveness. We find that IL1R2 is significantly upregulated at both the transcript and protein level in IL-33 activated ILC2s. We are also able to demonstrate that ADAM17 regulates IL1R2 levels on ILC2s in both a constitutive and activation induced manner. Additionally, IL1R2+ ILC2s, a unique subset of ILC2s, have decreased Il5 and Il13 transcripts following IL-33 stimulation. Overall, these data suggest that the expression of IL1R2 may act as an activation-induced negative regulatory feedback mechanism to decrease ILC2 responsiveness to IL-33.

ADAM10-Mediated ICOS Ligand Shedding on B Cells Is Necessary for Proper T Cell ICOS Regulation and T Follicular Helper Responses.

The proper regulation of ICOS and ICOS ligand (ICOSL) has been shown to be essential for maintaining proper immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant Ab production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that a disintegrin and metalloproteinase (ADAM)10 is the primary physiological sheddase of ICOSL in mice and humans. Using an in vivo system in which ADAM10 is deleted only on B cells, elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wild-type mice, interaction of ICOS/ICOSL results in ADAM10-induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper development and TH2 polarization, as seen in a house dust mite exposure model. In addition, enhanced TH1 and TH17 immune responses are seen in experimental autoimmune encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and rescues, at least in part, T follicular helper numbers and the abnormal Ab production previously reported in these mice. Overall, we propose a novel regulation of the ICOS/ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL, as well as indirectly regulating ICOS, thus controlling ICOS/ICOSL-dependent responses.

Cutting edge: mast cells critically augment myeloid-derived suppressor cell activity.

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (Kit(Wsh)(/)(Wsh)) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Adipose tissue-selective ablation of ADAM10 results in divergent metabolic phenotypes following long-term dietary manipulation.

A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10), is involved in several metabolic and inflammatory pathways. We speculated that ADAM10 plays a modulatory role in adipose tissue inflammation and metabolism. To this end, we studied adipose tissue-specific ADAM10 knock-out mice (aKO). While young, regular chow diet-fed aKO mice showed increased insulin sensitivity, following prolonged (33 weeks) high-fat diet (HFD) exposure, aKO mice developed obesity and insulin resistance. Compared to controls, aKO mice showed less inflammatory adipokine profile despite the significant increase in adiposity. In brown adipose tissue, aKO mice on HFD had changes in CD8+ T cell populations indicating a lesser inflammatory pattern. Following HFD, both aKO and control littermates demonstrated decreased adipose tissue pro-inflammatory macrophages, and increased anti-inflammatory accumulation, without differences between the genotypes. Collectively, our observations indicate that selective deletion of ADAM10 in adipocytes results in a mitigated inflammatory response, leading to increased insulin sensitivity in young mice fed with regular diet. This state of insulin sensitivity, following prolonged HFD, facilitates energy storage resulting in increased fat accumulation which ultimately leads to the development of a phenotype of obesity and insulin resistance. In conclusion, the data indicate that ADAM10 has a modulatory effect of inflammation and whole-body energy metabolism.

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture.

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.

Contribution of αß T cells to macrophage polarization and MSC recruitment and proliferation on titanium implants.

Physiochemical cues like topography and wettability can impact the inflammatory response and tissue integration after biomaterial implantation. T cells are essential for immunomodulation of innate immune cells and play an important role in the host response to biomaterial implantation. This study aimed to understand how CD4+ and CD8+ T cell subsets, members of the αß T cell family, polarize in response to smooth, rough, or rough-hydrophilic titanium (Ti) implants and whether their presence modulates immune cell crosstalk and mesenchymal stem cell (MSC) recruitment following biomaterial implantation. Post-implantation in mice, we found that CD4+ and CD8+ T cell subsets polarized differentially in response to modified Ti surfaces. Additionally, mice lacking αß T cells had significantly more pro-inflammatory macrophages, fewer anti-inflammatory macrophages, and reduced MSC recruitment in response to modified Ti post-implantation than αß T cell -competent mice. Our results demonstrate that T cell activation plays a significant role during the inflammatory response to implanted biomaterials, contributing to macrophage polarization and MSC recruitment and proliferation, and the absence of αß T cells compromises new bone formation at the implantation site. STATEMENT OF SIGNIFICANCE: T cells are essential for immunomodulation and play an important role in the host response to biomaterial implantation. Our results demonstrate that T cells actively participate during the inflammatory response to implanted biomaterials, controlling macrophage phenotype and recruitment of MSCs to the implantation site.